An efficient and high-throughput mutagenesis strategy is an integral part of protein structure/function studies, directed evolution experiments for the discovery of novel proteins, and optimization of genetic elements in synthetic biology systems. Among the methods for in vitro mutagenesis known in the art, none offers a convenient, efficient and high-throughput approach for creating an extensive, user-defined library of variants in which single or multiple mutations can be located at any position. For example, site-directed mutagenesis methods, such as Kunkel mutagenesis (Kunkel, 1985), QuikChange (QuikChange Site-Directed Mutagenesis Kit, Stratagene), and inverse PCR (Doming and Andrews, 2003), are low-throughput methods. Combined chain reaction requires specially designed sets of primers and cloning of PCR products (Hames et al., 2005; Bi and Stambrook, 1998). Creating mutations by gene synthesis is comparatively expensive and requires sub-cloning of DNA. Error-prone PCR suffers from mutational bias, the inability to define the mutational composition, and the inability to effectively cause most amino acid substitutions, which require two or three mutations in a single codon. Methods that rely on random DNA cleavage reagents or transposons for mutating short sequences of DNA suffer from complex procedures and the inability to target the mutations (Baldwin et al., 2008; Murakami et al., 2002; Liu and Cropp, 2012).
Kunkel mutagenesis is a site-directed method developed to introduce mutations by using a mutation-encoding oligonucleotide (oligo) that anneals to a single-stranded uracil-containing circular DNA template. T7 DNA polymerase and T4 ligase are used to complete synthesis of the mutated strand. Upon transformation of E. coli, the newly synthesized mutated strand survives to a higher extent than the uracil-containing template strand. While the initial Kunkel protocol described making single base substitutions (Kunkel, 1987), other researchers have adapted the method for creating site-saturation libraries in a single codon (Scholle et al., 2005; Weiss et al., 2000). The mutational efficiency of site-directed Kunkel mutagenesis is limited such that typically 50-70% of transformed colonies harbor the desired mutation, while the remainder harbor the wildtype sequence (Kunkel et al., 1987).
Existing methods for site-directed mutagenesis at multiple distal sites simultaneously either have complex and multi-step procedures or have not been demonstrated to be efficient enough for library construction (Bi and Stambrook, 1998; QuikChange Multi Site-Directed Mutagenesis Kit, Stratagene). In addition, the mutagenesis toolbox currently lacks a method for creating extensive DNA libraries with a researcher-defined mutational composition spanning across an entire gene. For example, until now there has been no efficient method to make a library comprising all 18,900 possible single codon substitutions of a 300 amino acid long protein, nor is there an efficient method to make a user-prescribed subset of only 2000 of these 18,900 mutations. Accordingly, current methods to make multiple mutations simultaneously suffer from complicated procedures or low efficiencies and methods for site-directed mutagenesis for creating single mutations suffer from sub-optimal efficiency and variable success.